The co-translational N-glycosylation of many important cell surface receptors, ion channels and lysosomal enzymes is essential for their correct folding, intracellular routing and function in the CNS and other mammalian cells. The vital importance of protein N-glycosylation in humans is emphasized by the clinical consequences of a series of inherited genetic defects in this pathway classified as Congenital Disorders of Glycosylation (CDG). While the enzymology and topology of the biosynthesis of Glc3Man9GlcNAc2-P-P-Dol, the oligosaccharyl donor, in the endoplasmic reticulum (ER) have been studied extensively, many gaps remain in the understanding of how the enzymes in this pathway are organized and regulated. This proposal describes further studies to learn more about the role of ER-associated proteins in the regulation of dolichyl phosphate (Dol-P) biosynthesis and recycling, and the transbilayer movement of Man-P-Dol. Three Specific Aims are planned using biochemical, genetic and immunochemical approaches designed: 1) To utilize a recently cloned cDNA to learn more about the long-chain c/s-isoprenyltransferase (c/s-IPTase) catalyzing the elongation stage in Dol-P biosynthesis by investigating its regulation and the nature of its association with potential binding partners in the ER;2) To utilize cloned cDNAs encoding dolichol kinase (DK) and Dol-P-P phosphatase, an ER enzyme with a lumenally-oriented active site, in in vivo and in vitro experiments aimed at elucidating their precise roles in the ote novo synthesis of Dol-P and the recycling of the glycosyl carrier lipid and 3) To purify, identify and characterize the ER protein(s) mediating the transverse diffusion of Man-P-Dol in mammalian cells ("flippase"). The information gained on the structure of the Man-P-Dol flippase will be relevant to related membrane proteins mediating the transbilayer movement of other dolichyl-P-(P)-saccharide intermediates in protein O-, C- and N-glycosylation, glycerophospholipids, glucosylceramide and glycosylphosphatidylinositol (GPI) anchor precursors and potential defects in patients with Congenital Disorders of Glycosylation (CDG).